Three-way contact analysis characterizes the higher order organization of the Tcra locus

The generation of highly diverse antigen receptors in T and B lymphocytes relies on V(D)J recombination. The enhancer E(α) has been implicated in regulating the accessibility of V(α) and J(α) genes through long-range interactions during rearrangements of the T-cell antigen receptor gene Tcra. However, direct evidence for E(α) physically mediating the interaction of V(α) and J(α) genes is still lacking. In this study, we utilized the 3C-HTGTS assay, a chromatin interaction technique based on 3C, to analyze the higher order chromatin structure of the Tcra locus. Our analysis revealed the presence of sufficient information in the 3C-HTGTS data to detect multiway contacts. Three-way contact analysis of the Tcra locus demonstrated the co-occurrence of the proximal J(α) genes, V(α) genes and E(α) in CD4(+)CD8(+) double-positive thymocytes. Notably, the INT2–TEAp loop emerged as a prominent structure likely to be responsible for bringing the proximal J(α) genes and the V(α) genes into proximity. Moreover, the enhancer E(α) utilizes this loop to establish physical proximity with the proximal V(α) gene region. This study provides insights into the higher order chromatin structure of the Tcra locus, shedding light on the spatial organization of chromatin and its impact on V(D)J recombination.